MOLECULAR REPRODUCTION AND DEVELOPMENT 31:195-199 (1992)
Insulin-Like Growth Factor-1 Stimulates Growth of Mouse Preimplantation Embryos In Vitro MARK B. HARVEY AND PETER L. KAYE Department of Physiology and Pharmacology, The University of Queensland, Brisbane, Queensland, Australia
ABSTRACT Because recent studies have particularly implicated the insulin growth factor family in early development, the effects of insulin-like growth factor (IGF-1) on the development of mouse embryos in vitro were investigated in detail. When added to the medium for culture of two-cell embryos, IGF-1 stimulated the number of cells in the resultant blastocysts after 54 hr, entirely by increasing the number of cells in the inner cell mass (ICM) (16.0 2 0.5 vs. 12.6 0.5 cells/lCM). This stimulation was also achieved when ICMs were isolated from blastocysts prior to culture for 24 hr with IGF-1 (22.3 2 1.0 vs. 17.5 L 0.8 cells/lCM). There was no effect of IGF-1 on trophectoderm (TE) cell proliferation. In morphology studies, IGF-1 also increased the proportion of blastocysts (62%2 3%vs. 49% 2 4%)while decreasing the number of embryos remaining as morulae (32%2 3%vs. 38% 2%) or in the early cleavage stages (7% 3% vs. 13% 2 3%) after 54 hr culture from the two-cell stage. All these effects were achieved with EC,,s of approximately 60 pM IGF-1, which is in the range for IGF-1 receptor mediation; however, cross reaction with insulin, IGF-2, or other unknown receptors is not excluded. Nonetheless, the results show that physiological concentrations of IGF-1 (17-170 pM, 0.1-1 ng/ml), which have been observed in the reproductive tract, affect the early embryo, suggesting a normal role for this factor in the regulation of growth of the developing conceptus before implantation.
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Key Words: IGF-1, Inner cell mass, Trophectoderm
INTRODUCTION Although preimplantation embryos develop normally in defined media (Biggers e t al., 1971), their cleavage rate is slower than that of those t h a t develop in vivo (Bowman and McLaren, 1970; Harlow and Quinn, 1982). This implies that the presence of growth factors in the reproductive tract may enable embryos to obtain optimum development not possible in a defined culture medium. Insulin has been shown to stimulate metabolism and growth of mouse embryos (Heyner et al., 1989; Harvey and Kaye, 1988,1990,1991a; Gardner and Kaye, 1991) via its own receptors, which are present from the compacted eight-cell stage until at least the late blastocyst stage (Rappolee et al., 1990; Harvey and Kaye, 1991b). Also, insulin’s related growth factor, insulin-like growth factor-1 (IGF-1), stimulates embryonic metabo-
0 1992 WILEY-LISS, INC.
lism (Rappolee et al., 1990; Harvey and Kaye, 1991a), and the receptor mRNA (Rappolee et al., 1990) and complex (Mattson et al., 1988) have been detected in early embryos. This study was aimed at investigating IGF-1’s growth effects on preimplantation mouse embryos to determine if the addition of IGF-1 to a standard culture medium could enhance the developmental potential of embryos developing in vitro.
MATERIALS AND METHODS Superovulation and Embryo Manipulation Randomly bred Quackenbush mice (10 weeks) were superovulated by intraperitoneal injections of 10 IU pregnant mare serum gonadotropin (PMSG) at 1000 h r followed 48 h r later by 10 IU human chorionic gonadotropin (hCG) (Folligon & Chorulon, Intervet, Australia) and paired with males. Mating was determined by the presence of a vaginal plug at 0900 h r the following morning. Two-cell embryos were collected 48 h r posthCG in M2 (Fulton and Whittingham, 1978; modified as previously described by Hobbs and Kaye, 1985) and cultured in groups of 20-30 in 30 pl droplets of BMOC2 (Brinster, 1965; modified as previously described by Pemble and Kaye, 1986) under liquid paraffin oil at 37°C in a humidified atmosphere of 5%CO,, 5% O,, and 90% N,. Effect of IGF-1 on Morphological Development and Cell Number Two-cell embryos were cultured for 54 hr, i.e., to 102 h r post-hCG, with various concentrations of IGF-1 (recombinant analogue of human somatomedin C; Amersham Pty Ltd, Australia) in BMOC2 before assessing morphological development and cell number. Embryos were classified as blastocysts (those embryos possessing a visible blastocoel); morulae; or cleavage-stage embryos, which were those that contained two or more cells but had not undergone compaction; i.e., no interblastomeric junctions were visible. The numbers of cells in the trophectoderm (TE) and inner cell mass (ICM) were counted after differential nuclear staining (Handyside and Hunter, 1984; modified as previously described by Harvey and Kaye, 1990). Received June 27,1991; accepted October 7, 1991. Address reprint requests to Peter L. Kaye, Department of Physiology and Pharmacology,The University of Queensland, Brisbane, Queensland, Australia 4072.
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M.B. HARVEY AND P.L. KAYE
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Fig. 1. Cell number of whole blastocysts, inner cell masses (ICM), and trophectoderm (TE)after 54 hr of culture from two-cell embryos in BMOCP (C) or BMOCZ + 1.7 nM IGF-1 (IGF-1). Means -+ SEMs of three experiments, each containing 10 blastocysts/treatment. *P < 0.05, ***P < 0.001 by ANOVA.
IGF-1 Effect on Isolated ICM The ICMs of blastocysts collected 96 h r post-hCG were isolated by immunosurgery (Solter and Knowles, 1975; modified as previously described by Harvey and Kaye, 1990) and cultured for 24 h r with various concentrations of IGF-1 in BMOC2 before determination of cell number using bisbenzimide (Hoechst 33258) (Harvey and Kaye, 1990).
Statistical Analysis Statistical analysis of cell number studies was by analysis of variance (ANOVA). The percentage of twocell embryos developing to the various morphological stages after 54 h r of culture was transformed to radians to normalise the results and analysed by two-way ANOVA for treatment and experimental effects. RESULTS Effect of IGF-1 on Mitogenesis There were 11%more cells in blastocysts that developed from two-cell embryos with 1.7 nM IGF-1. This was entirely due to a 27% increase in the number of cells in the ICM; IGF-1 had no effect on TE cell numbers (Fig. 1). Furthermore, when ICMs were isolated from 96 h r post-hCG blastocysts and cultured for a further 24 h r in vitro with 0 (17.5 * 0.8 cells), 0.17 nM (22.3 L 1.0 cells), or 1.7 nM (22.8 0.8 cells) IGF-1, there was a n identical =30% increase in cell numbers of those developing in either concentration of IGF-1 (P < 0.001; from four experiments each containing four to seven ICMs/ treatment). In the blastocysts that had developed in vitro, there appeared to be no evidence of a select group of IGF-1-responsive embryos; the frequency distributions of ICM cell numbers for both groups were not significantly different from normal (P > 0.9; Fig. 2) when tested with the Kolomogorov-Smirnov test (Pfaffenberger and Patterson, 1977).
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Fig. 2. Frequency distributions of ICM cell number in whole blastocysts from BMOCP medium (A) and BMOCP +1.7 nM IGF-1 (B). Means are indicated by arrows. Data are from Figure 1. The lines are the normal frequency distributions with means and SDs indicated in Figure 1.
Effect of IGF-1 on Morphological Development After 54 h r of culture from the two-cell stage with 1.7 nM IGF-1, 13% more blastocysts (62% t 3% vs. 49% 4%; P < 0.05) had developed, and 6% fewer morulae (32% 3% vs. 38% * 2%) and 6% fewer cleavage-stage embryos (7% * 3% vs. 13% * 3%; P < 0.05) remained than in medium lacking IGF-1 (Fig. 3). The two-way ANOVA revealed no significant experimental variation.
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Dose-Response Studies Studies showed, of these effects of IGF-1, a significant correlation (P < 0.05, r = 0.97, n = 4) between the log of the IGF-1 concentration and ICM cell number in blastocysts developing in vitro with 17 pM to 170 pM IGF-1 (EC,, = 60 pM) when these data were fitted by least-squares linear regression (Fig. 4). In morphological studies, there was significant positive correlation between the log of the IGF-1 concentration and the proportion of blastocysts developing ( P < 0.01, r = 0.99, n = 4), and negative correlations with the proportions of morulae (P < 0.05, r = -0.97, n = 4) and
IGF-1 STIMULATES EARLY EMBRYO GROWTH
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Fig. 3. Percentage of embryos developing to various morphological stages after 54 hr of culture from two-cell embryos in BMOCB (C) and BMOC2 + 1.7 nM IGF-1. Means ? SEMs of eight experiments, each containing at least 24 embryos/treatment. *P< 0.05 by two-way ANOVA after transformation to radians.
cleavage-stage embryos (P < 0.05, r = -0.98, n = 4) remaining after 54 h r in the same IGF-1 concentrations (Fig. 5). The ECSOscalculated from the regression lines were 62 pM, 58 pM, and 49 pM IGF-1 for blastocysts, morulae, and cleavage-stage embryos, respectively.
DISCUSSION After 54 h r in culture, blastocysts that developed in the presence of IGF-1 contained more cells than those developing in control medium (Fig. 1). This increased blastocyst cell number was due entirely to a n increase in the number of cells in the ICM (Fig. 1). Furthermore, there was no evidence of a subpopulation of embryos unresponsive to IGF-1 (Fig. 2). In that the TE completely surrounds the ICM in the blastocyst, the effects of IGF-1 on the ICM in intact blastocysts arise from exogenous IGF-1 binding to the TE. To investigate if IGF-1 has direct actions by binding to ICM receptors, ICMs were immunosurgically isolated and cultured with IGF-1. There was a n identical increase in cell number of isolated ICMs at both 170 pM and 1.7 nM IGF-1, which suggests that IGF-1 may stimulate ICM proliferation directly. A similar observation has been reported for insulin (Harvey and Kaye, 1990). Assay of the morphological development after 54 h r showed that supplementation of the medium with IGF-1 increased the proportion of blastocysts forming by a further 13% to 62% of the two-cell embryos placed in culture (Fig. 3). There was also a decrease in the proportion of cleavage-stage embryos, indicating that IGF-1 also stimulated compaction. In that compaction and blastocyst formation are functions of the outer or TE cells, these observations indicate that these outer cells respond to IGF-1 metabolically but not by increased rate of proliferation (Fig. 1). Insulin has similarly been shown to stimulate growth during culture (Harvey and Kaye, 1990; Gardner and Kaye, 19911,
197
with this morphogenic effect diminishing after 72 h r in culture (Gardner and Kaye, 1991). This is consistent with the inability of others to show that IGF-1 had any effect after 72 h r of culture from the two-cell stage (Paria and Dey, 19901, which may be because more complex conditions are required to support optimal development of blastocysts at implantation in vitro (Sellens and Sherman, 1980). These mitogenic and morphogenic effects of IGF-1 correlate with studies that showed binding to mouse blastocysts (Mattson et al., 1988) and stimulation of protein synthesis (Rappolee et al., 1990; Harvey and Kaye, 1991a). In another study, this growth factor increased incorporation of labelled precursors of protein, RNA, and DNA (Rao et al., 1990), but this stimulation was not statistically significant, possibly because of insufficient exposure to IGF-1 [only 1h r prior to labelling compared with 4 h r in the Harvey and Kaye (1991a) and Rappolee e t al. (1990) studies] or, alternatively, insufficient numbers of experiments to achieve statistical significance. High concentrations of IGF-1 should cross react with insulin receptors (Froesch and Zapf, 19851, which are potent stimulators of embryonic metabolism and growth (Heyner et al., 1989; Harvey and Kaye, l990,1991a), and this was not observed, suggesting some complicating factor in that study. To gain some insight into the identity of the receptor mediating IGF-1’s effects, dose-response studies were performed. In both ICM mitogenesis (Fig. 4) and morphology (Fig. 5) studies, a n EC50 of about 60 pM IGF-1 was obtained, suggesting that the one receptor type mediates both the morphological and mitogenic responses. This is similar to ECS0sof 100 pM and 200 pM IGF-1 for mitogenic stimulation of hepatoma and myeloid leukemic cells, respectively (Ponzio et al., 1988; Pepe et al., 1987). In the latter cells, the mitogenic effect arises from actions via IGF-1 receptors. Thus the concentrations that elicit the growth actions of IGF-1 on mouse embryos are in the range for mediation by the IGF-1 receptor in other cells and the mRNA for this receptor was observed in morulae and blastocysts (Rappolee et al., 1990). However, insulin stimulated growth of mouse embryos via very-high-sensitivity insulin receptors with EC50of 0.5 pM (Harvey and Kaye, 1990). In that IGF-1 can cross react with insulin receptors (Rechler and Nissley, 1985), i t is possible that IGF-1 acted through these receptors. To complicate this issue of receptor identity further, the stimulation of both compaction and blastocyst formation by IGF-1 (Fig. 4) differs from the result seen with very low doses of insulin, which similarly stimulated blastocyst formation but had no effect on compaction (Harvey and Kaye, 1990). This suggests the presence of a mechanism of response to IGF-1 in embryos prior to compaction. However, there is no other evidence for the presence of IGF-1 receptors prior to compaction (Rappolee e t al., 1990). The mRNA for IGF-2 receptors is expressed from the two-cell stage (Rappolee et al., 1990), and the receptor complex has been observed directly using immunohistochemistry
M.B. HARVEY AND P.L. KAYE
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Fig. 4. Dose response to IGF-1 of ICM cell number of blastocysts cultured 54 hr from two-cell embryos in BMOC2 containing various concentrations of IGF-1. Means +- SEMs of three experiments, each containing eight to ten ICMdtreatment. Solid line was fitted by least-squares linear regression.
stage of development. In the absence of appropriate blocking antibodies, it is difficult to see how this identity can be resolved. Nevertheless, the important finding from these studies is that physiological concentrations of IGF-1 stimulate preimplantation development even though the responsible receptor(s) have not been identified.
1.0,
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1, 0
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lo2
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IGF-I (pU) Fig, 5. Dose response of the percentage of two-cell embryos reaching morphological stages (converted to radians) to IGF-1 after 54 hr of culture in BMOC2 medium. Means i_ SEM, of at least three experiments, each containing a t least 24 embryos/treatment. Squares, blastocyst; triangles, morulae; circles, cleavage-stage embryos. Solid lines were fitted by least-squares linear regression as described in the text.
(Harvey and Kaye, 1991~).Since IGF-1 may bind to IGF-2 receptors (Rechler and Nissley, 19851, this heterologous binding may explain the response of cleavagestage embryos to IGF-1 and insulin (Gardner and Kaye, 1991). If so, the IGF-2 receptors appear to have a high sensitivity to IGF-1 evidenced by the low EC,, observed in this study. Although the polymerase chain reaction (PCR) technique used to demonstrate presence of mRNA for insulin, IGF-1, and IGF-2 receptors at various stages of preimplantation embryo development did not indicate presence of IGF-1 receptors prior to compaction (Rappolee et al., 1990), it is possible that a variant mRNA not detected by the technique is present a t this early
CONCLUSIONS Physiological concentrations of IGF-1 produced more blastocysts with more inner cells than in control medium after 54 h r of development from two-cell embryos in vitro (102 h r post-hCG). The mitogenic actions although confined to ICMs were apparent in whole blastocysts. The identity of the receptorh-eceptors mediating these effects is unclear because of the high sensitivities of both these responses to insulin and IGF-1. Thus further studies with others of this growth factor family may help to resolve the identity of receptors responding to IGFs. The presence of IGF-1 in the reproductive tract (Murphy e t al., 1987) and the low EC50 for IGF-1 suggest a normal function for this ligand in regulating growth of the early embryo to attain full developmental potential, no matter which receptor is mediating the response. ACKNOWLEDGMENTS We thank Kathryn Markham for expert technical assistance. This work was supported by a research project grant to P.L.K. and a Biomedical Research Scholarship to M.B.H. from the National Health and Medical Research Council of Australia. REFERENCES Biggers JD, Whitten WK, Whittingham DG (1971): The culture of mouse embryos in vitro. In JC Daniel (ed): “Methods in Mammalian Embryology.” San Francisco: W.H. Freeman and Co. pp. 86-116. Bowman P, McLaren A (1970): Cleavage rate of mouse embryos in vivo and in vitro. J Embryo1 Expl Morphol24:203-207. Brinster RL (1965): Studies on the development of mouse embryos in vitro. IV. Interaction of energy sources. J Reprod Fertil 10:227-240.
IGF-1 STIMULATES EARLY EMBRYO GROWTH Froesch ER, Zapf J (1985): Insulin-like growth and insulin: comparative aspects. Diabetologia 28:485-493. Fulton B, Whittingham D (1978): Activation of mammalian oocytes by intracellular injection of calcium. Nature 273:149-151. Gardner HG, Kaye PL (1991): Insulin stimulates mitosis and morphological development in mouse preimplantation embryos in vitro. Reprod Fertil Dev 3:19-92. Handyside AH, Hunter S (1984):A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes. J Exp Zoo1 31:429434. Harlow GM, Quinn P (1982):Development of preimplantation mouse embryos in vivo and in vitro. Aust J Biol Sci 35:187-193. Harvey MB, Kaye PL (1988): Insulin stimulates protein synthesis in compacted mouse embryos. Endocrinology 122:1182-1184. Harvey MB, Kaye PL (1990): Insulin stimulates mitogenesis of the inner cell mass and morphological development of mouse blastocysts. Development 110:963-967. Harvey MB, Kaye PL (1991a): Mouse blastocysts respond metabolically to short term stimulation by insulin and IGF-1 through the insulin receptor. Mol Reprod Dev 29:253-258. Harvey MB, Kaye PL (1991b): Visualisation of insulin receptors on mouse preembryos. Reprod Fertil Dev 3:9-15. Harvey MB, Kaye PL (1991~):IGF-2 receptors are first expressed at the two-cell stage of mouse development. Development 111:10571060. Heyner S, Rao LV, Jarett L, Smith RM (1989): Preimplantation mouse embryos internalise maternal insulin via receptor-mediated endocytosis: Pattern of uptake and functional correlations. Dev Biol i34:4a58. Hobbs JG, Kaye PL (1985): Glycine transport in mouse eggs and preimplantation embryos. J Reprod Fertil74:77-86. Mattson BM, Rosenblum IY, Smith RM, Heyner S (1988): Autoradiographic evidence for insulin and insulin-like growth factor binding to early mouse embryos. Diabetes 372585489.
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Murphy LJ,Murphy LC, Friesen HG (1987): Oestrogen induces insulin-like growth factor-1 expression in the rat uterus. Mol Endocrinol 1:445450. Paria BC, Dey SK (1990): Preimplantation embryo development in vitro: Cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci USA 87:47564760. Pemble LB, Kaye PL (1986):Whole protein uptake and metabolism by mouse blastocysts. J Reprod Fertil78:149-157. Pepe MG, Ginzton NH, Lee PDK, Hintz RL, Greenberg PL (1987): Receptor binding and mitogenic effects of insulin and insulin-like growth factor 1 and 2 for human myeloid leukemic cells. J Cell Physiol 133:219-227. Pfaffenberger RC, Patterson J H (1977): “Statistical Methods.” Homewood, IL: Richard D Irwin, Inc. Ponzio G, Contreres JO, Debant A, Baron V, Gautier N, DolaisKitabgi J, Rossi B (1988): Use ofan anti-insulin receptor antibody to discriminate between metabolic and mitogenic effects of insulin: Correlation with receptor autophosphorylation. EMBO J 7:41114117. Rao LV, Wikarczuk ML, Heyner S (1990): Functional roles of insulin and insulin-like growth factors in preimplantation mouse embryo development. In Vitro Cell Dev Biol26:1043-1048. Rappolee DA, Sturm KS, Schultz GA, Pedersen RA, Werb Z (1990): The expression of growth factor ligands and receptors in preimplantation mouse embryos. In S. Heyner and L. Wiley (eds): “Early Embryo Development and Paracrine Relationships.” A. R. Liss, New York: Wiley-Liss, pp 11-25. Rechler MM, Nissley SP (1985): The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol47:425442. Sellens MH, Sherman MI (1980): Effects of culture conditions on the developmental programme of mouse blastocysts. J Embryo1 Exp Morph 56:l-22. Solter D, Knowles BB (1975): Immunosurgery of mouse blastocyst. Proc Natl Acad Sci USA 725099-5102.
Insulin-Like Growth Factor-1 Stimulates Growth of Mouse Preimplantation Embryos In Vitro MARK B. HARVEY AND PETER L. KAYE Department of Physiology and Pharmacology, The University of Queensland, Brisbane, Queensland, Australia
ABSTRACT Because recent studies have particularly implicated the insulin growth factor family in early development, the effects of insulin-like growth factor (IGF-1) on the development of mouse embryos in vitro were investigated in detail. When added to the medium for culture of two-cell embryos, IGF-1 stimulated the number of cells in the resultant blastocysts after 54 hr, entirely by increasing the number of cells in the inner cell mass (ICM) (16.0 2 0.5 vs. 12.6 0.5 cells/lCM). This stimulation was also achieved when ICMs were isolated from blastocysts prior to culture for 24 hr with IGF-1 (22.3 2 1.0 vs. 17.5 L 0.8 cells/lCM). There was no effect of IGF-1 on trophectoderm (TE) cell proliferation. In morphology studies, IGF-1 also increased the proportion of blastocysts (62%2 3%vs. 49% 2 4%)while decreasing the number of embryos remaining as morulae (32%2 3%vs. 38% 2%) or in the early cleavage stages (7% 3% vs. 13% 2 3%) after 54 hr culture from the two-cell stage. All these effects were achieved with EC,,s of approximately 60 pM IGF-1, which is in the range for IGF-1 receptor mediation; however, cross reaction with insulin, IGF-2, or other unknown receptors is not excluded. Nonetheless, the results show that physiological concentrations of IGF-1 (17-170 pM, 0.1-1 ng/ml), which have been observed in the reproductive tract, affect the early embryo, suggesting a normal role for this factor in the regulation of growth of the developing conceptus before implantation.
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Key Words: IGF-1, Inner cell mass, Trophectoderm
INTRODUCTION Although preimplantation embryos develop normally in defined media (Biggers e t al., 1971), their cleavage rate is slower than that of those t h a t develop in vivo (Bowman and McLaren, 1970; Harlow and Quinn, 1982). This implies that the presence of growth factors in the reproductive tract may enable embryos to obtain optimum development not possible in a defined culture medium. Insulin has been shown to stimulate metabolism and growth of mouse embryos (Heyner et al., 1989; Harvey and Kaye, 1988,1990,1991a; Gardner and Kaye, 1991) via its own receptors, which are present from the compacted eight-cell stage until at least the late blastocyst stage (Rappolee et al., 1990; Harvey and Kaye, 1991b). Also, insulin’s related growth factor, insulin-like growth factor-1 (IGF-1), stimulates embryonic metabo-
0 1992 WILEY-LISS, INC.
lism (Rappolee et al., 1990; Harvey and Kaye, 1991a), and the receptor mRNA (Rappolee et al., 1990) and complex (Mattson et al., 1988) have been detected in early embryos. This study was aimed at investigating IGF-1’s growth effects on preimplantation mouse embryos to determine if the addition of IGF-1 to a standard culture medium could enhance the developmental potential of embryos developing in vitro.
MATERIALS AND METHODS Superovulation and Embryo Manipulation Randomly bred Quackenbush mice (10 weeks) were superovulated by intraperitoneal injections of 10 IU pregnant mare serum gonadotropin (PMSG) at 1000 h r followed 48 h r later by 10 IU human chorionic gonadotropin (hCG) (Folligon & Chorulon, Intervet, Australia) and paired with males. Mating was determined by the presence of a vaginal plug at 0900 h r the following morning. Two-cell embryos were collected 48 h r posthCG in M2 (Fulton and Whittingham, 1978; modified as previously described by Hobbs and Kaye, 1985) and cultured in groups of 20-30 in 30 pl droplets of BMOC2 (Brinster, 1965; modified as previously described by Pemble and Kaye, 1986) under liquid paraffin oil at 37°C in a humidified atmosphere of 5%CO,, 5% O,, and 90% N,. Effect of IGF-1 on Morphological Development and Cell Number Two-cell embryos were cultured for 54 hr, i.e., to 102 h r post-hCG, with various concentrations of IGF-1 (recombinant analogue of human somatomedin C; Amersham Pty Ltd, Australia) in BMOC2 before assessing morphological development and cell number. Embryos were classified as blastocysts (those embryos possessing a visible blastocoel); morulae; or cleavage-stage embryos, which were those that contained two or more cells but had not undergone compaction; i.e., no interblastomeric junctions were visible. The numbers of cells in the trophectoderm (TE) and inner cell mass (ICM) were counted after differential nuclear staining (Handyside and Hunter, 1984; modified as previously described by Harvey and Kaye, 1990). Received June 27,1991; accepted October 7, 1991. Address reprint requests to Peter L. Kaye, Department of Physiology and Pharmacology,The University of Queensland, Brisbane, Queensland, Australia 4072.
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Fig. 1. Cell number of whole blastocysts, inner cell masses (ICM), and trophectoderm (TE)after 54 hr of culture from two-cell embryos in BMOCP (C) or BMOCZ + 1.7 nM IGF-1 (IGF-1). Means -+ SEMs of three experiments, each containing 10 blastocysts/treatment. *P < 0.05, ***P < 0.001 by ANOVA.
IGF-1 Effect on Isolated ICM The ICMs of blastocysts collected 96 h r post-hCG were isolated by immunosurgery (Solter and Knowles, 1975; modified as previously described by Harvey and Kaye, 1990) and cultured for 24 h r with various concentrations of IGF-1 in BMOC2 before determination of cell number using bisbenzimide (Hoechst 33258) (Harvey and Kaye, 1990).
Statistical Analysis Statistical analysis of cell number studies was by analysis of variance (ANOVA). The percentage of twocell embryos developing to the various morphological stages after 54 h r of culture was transformed to radians to normalise the results and analysed by two-way ANOVA for treatment and experimental effects. RESULTS Effect of IGF-1 on Mitogenesis There were 11%more cells in blastocysts that developed from two-cell embryos with 1.7 nM IGF-1. This was entirely due to a 27% increase in the number of cells in the ICM; IGF-1 had no effect on TE cell numbers (Fig. 1). Furthermore, when ICMs were isolated from 96 h r post-hCG blastocysts and cultured for a further 24 h r in vitro with 0 (17.5 * 0.8 cells), 0.17 nM (22.3 L 1.0 cells), or 1.7 nM (22.8 0.8 cells) IGF-1, there was a n identical =30% increase in cell numbers of those developing in either concentration of IGF-1 (P < 0.001; from four experiments each containing four to seven ICMs/ treatment). In the blastocysts that had developed in vitro, there appeared to be no evidence of a select group of IGF-1-responsive embryos; the frequency distributions of ICM cell numbers for both groups were not significantly different from normal (P > 0.9; Fig. 2) when tested with the Kolomogorov-Smirnov test (Pfaffenberger and Patterson, 1977).
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Fig. 2. Frequency distributions of ICM cell number in whole blastocysts from BMOCP medium (A) and BMOCP +1.7 nM IGF-1 (B). Means are indicated by arrows. Data are from Figure 1. The lines are the normal frequency distributions with means and SDs indicated in Figure 1.
Effect of IGF-1 on Morphological Development After 54 h r of culture from the two-cell stage with 1.7 nM IGF-1, 13% more blastocysts (62% t 3% vs. 49% 4%; P < 0.05) had developed, and 6% fewer morulae (32% 3% vs. 38% * 2%) and 6% fewer cleavage-stage embryos (7% * 3% vs. 13% * 3%; P < 0.05) remained than in medium lacking IGF-1 (Fig. 3). The two-way ANOVA revealed no significant experimental variation.
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Dose-Response Studies Studies showed, of these effects of IGF-1, a significant correlation (P < 0.05, r = 0.97, n = 4) between the log of the IGF-1 concentration and ICM cell number in blastocysts developing in vitro with 17 pM to 170 pM IGF-1 (EC,, = 60 pM) when these data were fitted by least-squares linear regression (Fig. 4). In morphological studies, there was significant positive correlation between the log of the IGF-1 concentration and the proportion of blastocysts developing ( P < 0.01, r = 0.99, n = 4), and negative correlations with the proportions of morulae (P < 0.05, r = -0.97, n = 4) and
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Fig. 3. Percentage of embryos developing to various morphological stages after 54 hr of culture from two-cell embryos in BMOCB (C) and BMOC2 + 1.7 nM IGF-1. Means ? SEMs of eight experiments, each containing at least 24 embryos/treatment. *P< 0.05 by two-way ANOVA after transformation to radians.
cleavage-stage embryos (P < 0.05, r = -0.98, n = 4) remaining after 54 h r in the same IGF-1 concentrations (Fig. 5). The ECSOscalculated from the regression lines were 62 pM, 58 pM, and 49 pM IGF-1 for blastocysts, morulae, and cleavage-stage embryos, respectively.
DISCUSSION After 54 h r in culture, blastocysts that developed in the presence of IGF-1 contained more cells than those developing in control medium (Fig. 1). This increased blastocyst cell number was due entirely to a n increase in the number of cells in the ICM (Fig. 1). Furthermore, there was no evidence of a subpopulation of embryos unresponsive to IGF-1 (Fig. 2). In that the TE completely surrounds the ICM in the blastocyst, the effects of IGF-1 on the ICM in intact blastocysts arise from exogenous IGF-1 binding to the TE. To investigate if IGF-1 has direct actions by binding to ICM receptors, ICMs were immunosurgically isolated and cultured with IGF-1. There was a n identical increase in cell number of isolated ICMs at both 170 pM and 1.7 nM IGF-1, which suggests that IGF-1 may stimulate ICM proliferation directly. A similar observation has been reported for insulin (Harvey and Kaye, 1990). Assay of the morphological development after 54 h r showed that supplementation of the medium with IGF-1 increased the proportion of blastocysts forming by a further 13% to 62% of the two-cell embryos placed in culture (Fig. 3). There was also a decrease in the proportion of cleavage-stage embryos, indicating that IGF-1 also stimulated compaction. In that compaction and blastocyst formation are functions of the outer or TE cells, these observations indicate that these outer cells respond to IGF-1 metabolically but not by increased rate of proliferation (Fig. 1). Insulin has similarly been shown to stimulate growth during culture (Harvey and Kaye, 1990; Gardner and Kaye, 19911,
197
with this morphogenic effect diminishing after 72 h r in culture (Gardner and Kaye, 1991). This is consistent with the inability of others to show that IGF-1 had any effect after 72 h r of culture from the two-cell stage (Paria and Dey, 19901, which may be because more complex conditions are required to support optimal development of blastocysts at implantation in vitro (Sellens and Sherman, 1980). These mitogenic and morphogenic effects of IGF-1 correlate with studies that showed binding to mouse blastocysts (Mattson et al., 1988) and stimulation of protein synthesis (Rappolee et al., 1990; Harvey and Kaye, 1991a). In another study, this growth factor increased incorporation of labelled precursors of protein, RNA, and DNA (Rao et al., 1990), but this stimulation was not statistically significant, possibly because of insufficient exposure to IGF-1 [only 1h r prior to labelling compared with 4 h r in the Harvey and Kaye (1991a) and Rappolee e t al. (1990) studies] or, alternatively, insufficient numbers of experiments to achieve statistical significance. High concentrations of IGF-1 should cross react with insulin receptors (Froesch and Zapf, 19851, which are potent stimulators of embryonic metabolism and growth (Heyner et al., 1989; Harvey and Kaye, l990,1991a), and this was not observed, suggesting some complicating factor in that study. To gain some insight into the identity of the receptor mediating IGF-1’s effects, dose-response studies were performed. In both ICM mitogenesis (Fig. 4) and morphology (Fig. 5) studies, a n EC50 of about 60 pM IGF-1 was obtained, suggesting that the one receptor type mediates both the morphological and mitogenic responses. This is similar to ECS0sof 100 pM and 200 pM IGF-1 for mitogenic stimulation of hepatoma and myeloid leukemic cells, respectively (Ponzio et al., 1988; Pepe et al., 1987). In the latter cells, the mitogenic effect arises from actions via IGF-1 receptors. Thus the concentrations that elicit the growth actions of IGF-1 on mouse embryos are in the range for mediation by the IGF-1 receptor in other cells and the mRNA for this receptor was observed in morulae and blastocysts (Rappolee et al., 1990). However, insulin stimulated growth of mouse embryos via very-high-sensitivity insulin receptors with EC50of 0.5 pM (Harvey and Kaye, 1990). In that IGF-1 can cross react with insulin receptors (Rechler and Nissley, 1985), i t is possible that IGF-1 acted through these receptors. To complicate this issue of receptor identity further, the stimulation of both compaction and blastocyst formation by IGF-1 (Fig. 4) differs from the result seen with very low doses of insulin, which similarly stimulated blastocyst formation but had no effect on compaction (Harvey and Kaye, 1990). This suggests the presence of a mechanism of response to IGF-1 in embryos prior to compaction. However, there is no other evidence for the presence of IGF-1 receptors prior to compaction (Rappolee e t al., 1990). The mRNA for IGF-2 receptors is expressed from the two-cell stage (Rappolee et al., 1990), and the receptor complex has been observed directly using immunohistochemistry
M.B. HARVEY AND P.L. KAYE
198
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Fig. 4. Dose response to IGF-1 of ICM cell number of blastocysts cultured 54 hr from two-cell embryos in BMOC2 containing various concentrations of IGF-1. Means +- SEMs of three experiments, each containing eight to ten ICMdtreatment. Solid line was fitted by least-squares linear regression.
stage of development. In the absence of appropriate blocking antibodies, it is difficult to see how this identity can be resolved. Nevertheless, the important finding from these studies is that physiological concentrations of IGF-1 stimulate preimplantation development even though the responsible receptor(s) have not been identified.
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IGF-I (pU) Fig, 5. Dose response of the percentage of two-cell embryos reaching morphological stages (converted to radians) to IGF-1 after 54 hr of culture in BMOC2 medium. Means i_ SEM, of at least three experiments, each containing a t least 24 embryos/treatment. Squares, blastocyst; triangles, morulae; circles, cleavage-stage embryos. Solid lines were fitted by least-squares linear regression as described in the text.
(Harvey and Kaye, 1991~).Since IGF-1 may bind to IGF-2 receptors (Rechler and Nissley, 19851, this heterologous binding may explain the response of cleavagestage embryos to IGF-1 and insulin (Gardner and Kaye, 1991). If so, the IGF-2 receptors appear to have a high sensitivity to IGF-1 evidenced by the low EC,, observed in this study. Although the polymerase chain reaction (PCR) technique used to demonstrate presence of mRNA for insulin, IGF-1, and IGF-2 receptors at various stages of preimplantation embryo development did not indicate presence of IGF-1 receptors prior to compaction (Rappolee et al., 1990), it is possible that a variant mRNA not detected by the technique is present a t this early
CONCLUSIONS Physiological concentrations of IGF-1 produced more blastocysts with more inner cells than in control medium after 54 h r of development from two-cell embryos in vitro (102 h r post-hCG). The mitogenic actions although confined to ICMs were apparent in whole blastocysts. The identity of the receptorh-eceptors mediating these effects is unclear because of the high sensitivities of both these responses to insulin and IGF-1. Thus further studies with others of this growth factor family may help to resolve the identity of receptors responding to IGFs. The presence of IGF-1 in the reproductive tract (Murphy e t al., 1987) and the low EC50 for IGF-1 suggest a normal function for this ligand in regulating growth of the early embryo to attain full developmental potential, no matter which receptor is mediating the response. ACKNOWLEDGMENTS We thank Kathryn Markham for expert technical assistance. This work was supported by a research project grant to P.L.K. and a Biomedical Research Scholarship to M.B.H. from the National Health and Medical Research Council of Australia. REFERENCES Biggers JD, Whitten WK, Whittingham DG (1971): The culture of mouse embryos in vitro. In JC Daniel (ed): “Methods in Mammalian Embryology.” San Francisco: W.H. Freeman and Co. pp. 86-116. Bowman P, McLaren A (1970): Cleavage rate of mouse embryos in vivo and in vitro. J Embryo1 Expl Morphol24:203-207. Brinster RL (1965): Studies on the development of mouse embryos in vitro. IV. Interaction of energy sources. J Reprod Fertil 10:227-240.
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Murphy LJ,Murphy LC, Friesen HG (1987): Oestrogen induces insulin-like growth factor-1 expression in the rat uterus. Mol Endocrinol 1:445450. Paria BC, Dey SK (1990): Preimplantation embryo development in vitro: Cooperative interactions among embryos and role of growth factors. Proc Natl Acad Sci USA 87:47564760. Pemble LB, Kaye PL (1986):Whole protein uptake and metabolism by mouse blastocysts. J Reprod Fertil78:149-157. Pepe MG, Ginzton NH, Lee PDK, Hintz RL, Greenberg PL (1987): Receptor binding and mitogenic effects of insulin and insulin-like growth factor 1 and 2 for human myeloid leukemic cells. J Cell Physiol 133:219-227. Pfaffenberger RC, Patterson J H (1977): “Statistical Methods.” Homewood, IL: Richard D Irwin, Inc. Ponzio G, Contreres JO, Debant A, Baron V, Gautier N, DolaisKitabgi J, Rossi B (1988): Use ofan anti-insulin receptor antibody to discriminate between metabolic and mitogenic effects of insulin: Correlation with receptor autophosphorylation. EMBO J 7:41114117. Rao LV, Wikarczuk ML, Heyner S (1990): Functional roles of insulin and insulin-like growth factors in preimplantation mouse embryo development. In Vitro Cell Dev Biol26:1043-1048. Rappolee DA, Sturm KS, Schultz GA, Pedersen RA, Werb Z (1990): The expression of growth factor ligands and receptors in preimplantation mouse embryos. In S. Heyner and L. Wiley (eds): “Early Embryo Development and Paracrine Relationships.” A. R. Liss, New York: Wiley-Liss, pp 11-25. Rechler MM, Nissley SP (1985): The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol47:425442. Sellens MH, Sherman MI (1980): Effects of culture conditions on the developmental programme of mouse blastocysts. J Embryo1 Exp Morph 56:l-22. Solter D, Knowles BB (1975): Immunosurgery of mouse blastocyst. Proc Natl Acad Sci USA 725099-5102.
